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Changes of secondary metabolites in Pinus sylvestris L.
needles under increasing soil water deficit
Domingo Sancho-Knapik, María Ángeles Sanz, José Javier Peguero-Pina, Ülo
Niinemets, Eustaquio Gil-Pelegrín
To cite this version:
ORIGINAL PAPER
Changes of secondary metabolites in Pinus sylvestris L. needles
under increasing soil water deficit
Domingo Sancho-Knapik1,2&María Ángeles Sanz3&José Javier Peguero-Pina1,2&
Ülo Niinemets4&Eustaquio Gil-Pelegrín1,2
Received: 13 September 2016 / Accepted: 31 January 2017 / Published online: 8 March 2017 # INRA and Springer-Verlag France 2017
Abstract
&Key message A multiphasic response to water deficit was found in Scots pine primary and secondary metabolism. First, an increase of terpenoids coincided with the stomatal closure. Second, an accumulation of proline, ABA, and shikimic acid was detected when photosynthesis was negligible.
&Context Drought-induced mortality is characterized by a major needle yellowing followed by severe defoliation and whole branch death. Before these external visual symptoms of drought stress take place, different alterations occur in plant metabolism.
&Aims This study aims to detect changes in primary and sec-ondary metabolism of Pinus sylvestris L. in response to a decrease in soil water availability.
&Methods We analyzed needle water potential, photosynthet-ic characteristphotosynthet-ics, and concentrations of proline, terpenoids, shikimic acid, total polyphenols, and abscisic acid (ABA) in P. sylvestris through a 55-day soil water deficit period.
&Results Concentrations of most metabolites varied with the decrease in soil water availability, but changes in different compounds were triggered at different times, highlighting a multiphasic response. Increases in monoterpene and sesquiterpenoid content at moderate water deficit coincided
Handling Editor: Andrew Merchant
Contribution of the co-authors DSK, MAS, JJPP, ÜN, and EGP conceived the study and participated in its design. MAS led the compound extraction analysis while DSK, JJPP, and EGP led the physiological measurements. DSK and EGP ran the data analysis. DSK drafted the manuscript, and MAS, JJPP, ÜN, and EGP critically revised the manuscript.
* Eustaquio Gil-Pelegrín [email protected] Domingo Sancho-Knapik [email protected] María Ángeles Sanz [email protected] José Javier Peguero-Pina [email protected] Ülo Niinemets [email protected]
1
Unidad de Recursos Forestales, Gobierno de Aragón, Centro de Investigación y Tecnología Agroalimentaria, 50059 Zaragoza, Spain
2 Instituto Agroalimentario de Aragón (IA2), CITA, Universidad de
Zaragoza, Zaragoza, Spain
3
Área de Laboratorios de Análisis y Asistencia Tecnológica, Gobierno de Aragón, Centro de Investigación y Tecnología Agroalimentaria, 50059 Zaragoza, Spain
4
Institute of Agricultural and Environmental Sciences, Estonian University of Life Sciences, Kreutzwaldi1, 51014 Tartu, Estonia
with stomatal closure which preceded the accumulation of proline, ABA, and shikimic acid under severe water deficit when net photosynthesis was negligible.
& Conclusion This work confirms that most of the secondary metabolites under investigation in Pinus sylvestris did not in-crease until a moderate to severe water deficit was experi-enced, when photosynthesis was limited by stomatal closure.
Keywords Abscisic acid . Gas exchange . Shikimic acid . Water deficit . Proline . Terpenoids . Water potential
1 Introduction
Tree populations are facing new and rapidly changing selec-tive pressures such as more frequent and more severe droughts, threatening the conservation of forests and related ecosystem services (Lindner et al.2010; Martínez-Vilalta et al.
2012). Northern hemisphere conifer Pinus sylvestris L. has an exceptionally wide range of dispersal from subarctic to close to Mediterranean and warm temperate ecosystems, extending to its southern distribution limit to different mountain ranges of the Iberian Peninsula (Matías and Jump2012). In these southernmost habitats, this species appears sensitive to an in-crease in the aridity, as indicated by major tree deaths during severe drought episodes (Galiano et al.2010; Peguero-Pina et al. 2011; Sanz et al. 2014). The onset of drought-dependent mortality is visible by general needle yellowing followed by severe defoliation and whole branch death in a large fraction of trees (Sanz et al.2014).
Before these external visual symptoms of water deficit take place, numerous alterations in plant metabolism occur, includ-ing photosynthesis limitation, alteration of carbon allocation (Teskey et al.1987), changes in nutrient uptake, and variations in the levels of soluble sugars, inorganic ions, and amino acids (Schulze1991; Turtola et al.2003). Most recent studies on water deficit effects have focused on photosynthetic metabo-lism (e.g., Niinemets and Keenan2014); however, water def-icit also leads to modifications in secondary metabolism, and these changes often have remained hidden, but might provide a better insight into species and genotype variations in drought responses and into fine-tuning of plant functioning at a given level of primary metabolism (Niinemets2016).
As key changes under water deficit, several authors have re-ported accumulation of proline, which has been considered as a mechanism of osmotic adjustment helping plants to extract water from drier soils (Xiao et al.2008; Verbruggen and Hermans2008; Vilagrosa et al.2010). However, changes in secondary metabo-lism should not be directly related to adjustments to water deficit, but to other co-occurring stresses that can threaten the weakened plants, including adjustment to potentially more severe herbivory and pathogen infections. In conifers, it has been demonstrated that water deficit can modulate the composition of the oleoresin, changing the terpenoid profiles (monoterpenes, sesquiterpenoids, and diterpenoids, in particular, resin acids) (Lluisà and Peñuelas
1998; Turtola et al.2003). Moderate water deficit typically results in increased woody and needle terpene concentrations (Lluisà and Peñuelas1998; Turtola et al.2003), but severe water deficit can reduce terpene contents (Yani et al.1993). The role of terpenoids in the resistance or susceptibility of trees to attacks by diseases, in-sects, and animals and the influence of water deficit on such attacks
have been studied (Lluisà and Peñuelas1998; Pureswaran et al.
2004; Moreira et al.2009; Karanikas et al.2010), but the effects of water deficit severity on terpenoid content and composition and the corresponding effects on plant-insect and plant-pathogen in-teractions are poorly understood. Apart from terpenoids, phenolics and lignin are also involved in plant defense (Gamir et al.2014). These compounds derived from shikimic acid (Schafellner et al.
1999; Gamir et al.2014), the contents of which also seem to increase under water deficit (Becerra-Moreno et al.2015). In ad-dition, the accumulation of other compounds such as abscisic acid (ABA) has also been related to water deficit (Munné-Bosch et al.
2009; Corcuera et al.2012). Increase of ABA in water-deficient plants induces stomatal closure and reduces leaf growth (Tardieu et al.1992; Brodribb and McAdam2013), allowing to fine-tune the stomatal responses to water deficit.
Although changes in all of the abovementioned com-pounds have been related to water deficit, in most of the stud-ies, only two situations have been considered—well-watered and plants subjected to water deficit—and they fail to inves-tigate the metabolic transition through the development of the stress treatment. Moreover, the apparent contradictory results found in different papers about the effect of water deficit on the profile of some secondary compounds may be explained by the lack of a comparable experimental framework. In such a case, the interaction between water conservation and net CO2uptake as water deficit increases recommends the
simul-taneous measurement of physiological parameters and sec-ondary metabolites in order to establish a correspondence be-tween them. For these reasons, the main objective of this study was to monitor changes in content of all the key metabolites through mild to severe soil water deficit together with primary plant physiological characteristics—water potential, stomatal conductance, and net CO2uptake. More specifically, we have
analyzed the contents of proline, terpenoids (monoterpenes, sesquiterpenoids, and resin acids), shikimic acid, total poly-phenols, and ABA during a soil water deficit period in P. sylvestris, a species particularly threatened by global change. We hypothesized that the plant response as soil water deficit increases may reflect a timetable of coordinated events between primary and secondary metabolism, which could re-veal the physiological thresholds behind major changes in secondary metabolism. Understanding such coordinated changes will provide a framework to diagnosing the deficit situation in plants before external visual symptoms of drought stress become perceptible.
2 Materials and methods
2.1 Plant material and experimental conditions
were sown and cultivated in 2000 (mixture of 80% substratum and 20% perlite in 500-mL containers) inside a greenhouse. After the first growth cycle, seedlings were transplanted to 10-L containers, and after 2 years, they were again transplanted to 50-L containers (80% substratum, 20% perlite) and cultivated in a common garden (41°39′N, 0°52′W, Zaragoza, Spain; mean annual temperature 15.4 °C, total annual precipitation 298 mm). All plants were grown under the same environmen-tal conditions and irrigated every 3 days.
On August 2012, eight trees (12 years old) were moved from outside into a greenhouse (mean daily temperature 18.1 ± 0.5 °C, mean daily relative humidity 77.6 ± 1.1%). Once there, trees were watered every second day to field ca-pacity until September 12, 2012. At this date, watering of five experimental trees was stopped, while the other three trees were kept well-watered during the experiment as control trees. Soil water deficit was imposed for 55 days, and during the treatment, measurements of water potential and gas exchange were conducted periodically as the severity of water deficit increased. Measurements of water potentials were conducted at predawn while measurements of needle gas exchange were made at 8:00 h solar time (early morning). In addition, current-year needle samples for metabolite measurements in experi-mental and control trees were collected at predawn and stored at−80 °C for sample preservation until analysis.
2.2 Water potential and gas exchange measurements Predawn water potential (Ψpd, MPa) was measured in shoots
of P. sylvestris with a Scholander pressure chamber following the methodological procedure described by Turner (1988). Net CO2uptake (A,μmol CO2m−2s−1) and stomatal
conduc-tance (gs, mmol H2O m−2s−1) were measured in needles with
a portable gas exchange system (CIRAS-2, PP Systems, Herts, UK). Measurements were performed at controlled cu-vette CO2concentration (Ca) of 400μmol mol−1,
photosyn-thetic quantum flux density incident on the leaf surface of 1300μmol photons m−2s−1, and ambient relative humidity. 2.3 Analysis of monoterpenes and sesquiterpenoids and resin acid contents
For monoterpene and sesquiterpenoid analysis, frozen needles were cut into small pieces, introduced in vial tubes, and ex-tracted twice with n-hexane at room temperature for 2 h. The extracts were filtered (PTFE 0.20μm), and 1 μL was injected in a gas chromatography-mass spectrometer (GC-MS) (Agilent Technologies GC 6890 Series, MSD 5973N). 1-Chlorooctane was added as an internal standard.
Resin acids were extracted from freeze-dried and powdered needles, basically following the methodology of Gref and Ericsson (1985). One hundred milligrams was extracted with diethyl ether/petroleum ether (1:1, v/v) and twice with diethyl
ether. Supernatants were combined and evaporated to dryness under a stream of nitrogen. The residue was redissolved in 1 mL of diethyl ether and filtered (0.22 μm Nylon filter). Heptadecanoic acid was used as an internal standard. An ali-quot of extract was dried again before derivatization that was performed with N,O-bis-(trimethylsilyl)-trifluoroacetamide with trimethylchlorosilane (BSTFA-TMS)/acetonitrile (1:1; v/v) (90 min, 75 °C). One microliter of the solution was injected in the GC-MS.
Separation of the compounds was carried out with a HP-5MS column (30 m, 0.25 mm, 0.25μm). The temperature pro-gram for monoterpenes and sesquiterpenoids was 60 °C isother-mal 1 min, increased to 246 °C at 3 °C min−1. For resin acids, the temperature program was 60 °C isothermal 2 min, increased to 270 °C at 5 °C min−1. Helium was used as a carrier gas. Injector temperature was 220 °C in split mode (ratio 1:10). The transfer line temperature was 240 °C. The mass selective detector was operated in electron impact mode at 70 eV. MS source and MS quad temperatures were 230 and 150 °C, respectively, for monoterpenes and sesquiterpenoids and 250 and 200 °C, re-spectively, for resin acids. An MSD ChemStation (ver. E.02.00) workstation was used for data capture and processing with Scan and the selected ion monitoring (SIM) techniques. The identification of the chemical constituents was based on comparisons of their relative retention times and mass spectra with those obtained from authentic standards and/or mass spec-tra in NIST MS search 2.0 and Wiley 275 libraries. Further, the Kovats index (KI) was calculated using a homologous series on n-alkanes (Adams2009).
2.4 Abscisic acid determination
Fifty milligrams of lyophilized tissue was extracted twice with 3 mL of acetone/water/formic acid (80:19:1, v/v/v) (30 min, 2000 rpm) and centrifuged (15 min, 3000 rpm, 4 °C). The acetone was evaporated under a nitrogen stream, and the re-maining aqueous extract was adjusted to 1.2 mL with Milli-Q water. The extract was partitioned twice with diethyl ether, dried under nitrogen, and redissolved in 500μL acetonitrile/ water (30:70, v/v) containing 0.1% formic acid. The extract was filtered (0.45-μm Nylon filter) and diluted (1:5) before the UPLC system injection (ACQUITY, Waters). One nano-grams per microliter of [2H6]-ABA (Gómez-Cadenas et al.
2002) was added as an internal standard.
An Excel 2 C18-AR column (50 × 2.1 mm, ACE, UK) was used, and the temperature was set at 36 °C. The mobile phase was composed of methanol 70% (solvent A) and acetonitrile 90% (solvent B) which contain 0.1% formic acid. The solvent gradient was programmed to change linearly: 0–1 min, 100% A; 1–2.5 min, 100–50% A; 2.5–2.8 min, 50% A; and 2.8– 3 min, 100% A, for 2 min equilibration prior to the next injection. The solvent flow rate and injection volume were set at 0.15 mL min−1and 20μL, respectively.
Mass spectrometric analyses were performed by an ACQUITY Waters triple quadrupole mass spectrometer. The electrospray conditions were as follows: Polarity ES, capillary voltage 3.0 kV, source temperature 120 °C, desolvation gas temperature 350 °C, cone gas flow 90 L h−1, and desolvation gas flow 900 L h−1. High-purity nitrogen was used as the nebulizer and auxiliary gas, and argon was used as the colli-sion gas. Analysis of ABA was based on appropriate multiple reaction monitoring (MRM) mode: [2H6]ABA and ABA were
monitored at m/z transitions of 269→ 159, 225, and 263 → 153, 219, respectively. The cone voltage (V) and collision energies (eV) were optimized to maximize the transition sig-nals: [2H6]ABA 20 V and 12 eV, and ABA 15 V, 10 eV, and
12 eV. The raw data were acquired and processed with a MassLynx 4.1 software. Quantification was performed using calibration curves based on the ABA/[2H6]ABA ratio of
stan-dard solutions.
2.5 Quantification of proline, shikimic acid, and total polyphenols
Needle proline contents were determined following the proce-dure of Bates et al. (1973). Fifty milligrams of lyophilized tissue was processed with 3% (w/v) sulfosalicylic acid, and an aliquot of the supernatant was colored by the addition of ninhydrin. The heated ninhydrin-proline complex (100 °C, 1 h) was mixed with 4 mL of toluene. The condensation prod-uct was measured spectrophotometrically at 520 nm (Shimadzu UV-1700). The proline concentration was calcu-lated using aL-proline standard curve.
Shikimic acid was determined together with resin acids via the GC-MS analyses. Total polyphenol content was estimated in an aliquot of the extracts obtained to determine ABA. Two hundred milliliters was diluted with Milli-Q water and mixed with Folin-Ciocalteau reagent. The absorbance was measured at 725 nm (Velioglu et al.1998). Results were expressed as milligrams of gallic acid equivalents (GAEs) per gram dry weight.
2.6 Determination of physiological phases
Three physiological phases (I, II, and III) were established to analyze the metabolic compounds extracted from the needles. To define the limits of each phase, we took the points of physiological change of the relationships betweenΨpdand
gas exchange (gs and A). The first limit (Ψpd,1), between
phases I and II, was defined as the change in the slope of the relationship betweenΨpdand gs.Ψpd,1was calculated as the
join-point of a two-linear segmented model. This model is a non-linear model that fits a curve compound of two linear models with different slopes. The point at which the switch between the two functions occurs is generally called a joint-point, which can easily be associated with a change in the trend of the studied variable (Schabenberger and Pierce
2002). The second limit (Ψpd,2) was defined as the point where
A reached first time a value of zero since the application of the water deficit treatment. Ψpd,2was calculated from a linear
model adjusted to the relationship betweenΨpdand A.
2.7 Statistical analyses
While the contents of proline, shikimic acid, and ABA were related directly toΨpd, terpenoid contents were grouped
tak-ing into account the delineation of the three phases. One-way ANOVAs were performed to compare the contents of the met-abolic compounds between the different physiological phases (I, II, and III). Multiple comparisons were carried out among the different phases using the post hoc Tukey’s honestly sig-nificant difference test. For a more concise view, a principal component analysis (PCA) was made with those metabolic compounds that changed statistically along the soil water def-icit period. Data was expressed as means ± standard error. Statistical analyses were carried out using SAS version 8.0 (SAS, Cary, NC, USA), and all statistical analyses were con-sidered significant at P < 0.05.
3 Results
3.1 Physiological phases of water deficit development
The relationship between predawn water potential (Ψpd) and
stomatal conductance (gs), fitted by a two-linear segmented
model (R2 = 0.92, P < 0.001), had a joint-point at ca. −0.6 MPa (Fig.1a). For theΨpdrange of 0 to−0.6 MPa, gs
steeply decreased from maximum values of 500 mmol H2O m−2s−1to values around 150 mmol H2O m−2s−1. For
Ψpdvalues lower than−0.6 MPa, gsdecreased with a smaller
slope towards values of 50–20 mmol H2O m−2s−1. The linear
relationship between net CO2uptake (A) andΨpd(R2= 0.87,
P < 0.001) (Fig. 1b) showed a constant decrease in A from maximum values around 12μmol CO2m−2s−1to zero value
at ca.−1.8 MPa. Thus, Ψpdand gas exchange characteristics
suggested the existence of two key physiological modifica-tions along the decrease in soil water availability in P. sylvestris. The first one was atΨpd,1=−0.6 MPa, at which
point the rate of decrease in gs withΨpdchanged, and the
second one was atΨpd,2=−1.8 MPa, where photosynthesis
reached values of zero. These two criteria were used to define the three physiological phases of soil water deficit develop-ment: phase I (Ψpd>−0.6 MPa), denoting a mild water deficit;
phase II (−0.6 > Ψpd>−1.8 MPa), denoting a moderate water
deficit with significantly reduced values of gs and A; and
phase III (−1.8 > Ψpd>−2.4 MPa), denoting a severe water
deficit with completely suppressed photosynthesis. For the control trees, the average (± SE) values of Ψpd, gs, and A
397 ± 15 mmol H2O m−2 s−1, and 11.1 ± 0.2 μmol
CO2m−2s−1, respectively.
3.2 Modification of needle secondary metabolism through the water deficit phases
Average contents of most monoterpenes and sesquiterpenoids increased with increasing the severity of water deficit (Table1), except for a few compounds (e.g., sabinene or γ-muurolene) that did not vary significantly among the phases (Table 1). The statistical analysis revealed significant
differences in the content of volatile terpenes (e.g.,α-pinene, limonene, orβ-caryophyllene) already between phases I and II (P < 0.05). In contrast, the content of resin acids tended to decrease throughout the phases (Table1). This apparent de-crease was statistically significant (P < 0.05) for three of the seven resin acids analyzed (palustric, levopimaric, and neoabietic acids). For all terpenoids, mean values of control trees did not differ significantly from the averages of the water-deficient trees at phase I (Table1).
During water deficit phases I and II, proline and shikimic acid remained practically constant with mean values around 0.35μmolg−1and 0.82 mg g−1, respectively. In phase III, needle proline and shikimic acid content rose up to mean values of 1.24 ± 0.06μmol g−1and 2.40 ± 0.12 mg g−1respectively, i.e., ca. threefold higher than the mean values for phases I and II (Fig.2a, b). There was no significant effect of soil water deficit treatment on total polyphenol content (Fig.2b). Abscisic acid (ABA) content was statistically invariable (around 1.0μg g−1) for aΨpdrange of 0 to−1.3 MPa. Beyond −1.3 MPa, ABA
content started to increase exponentially (Fig.2c). ABA content reached at the end of phase II 2.8 ± 0.5μg g−1and in phase III 11.7 ± 0.0μg g−1(both statistically different from the initial values, P < 0.05). Control trees had mean values of proline, shikimic acid, and ABA of 0.38 ± 0.02 μmol g−1, 0.61 ± 0.07 mg g−1, and 0.73 ± 0.15μg g−1, respectively, through the experimental period. These control values were not statistically different from the mean values of water-deficient trees at phases I and II, but they were significantly lower than those at phase III (P < 0.05).
A PCA based on those compounds that changed statistical-ly along the water deficit period (monoterpenes and sesquiterpenoids, proline, shikimic acid, and ABA) indicated that the first and second axes accounted for 56 and 29% of the total variation, respectively (Fig.3). While we detected a seg-regation between the three physiological phases established (I–III), values of control trees were not segregated from the values of phase I (Fig.3).
4 Discussion
4.1 Soil water deficit severity in Pinus sylvestris: mild water deficit primarily alters photosynthetic
characteristics
Modifications in needle predawn water potential and gas exchange characteristics defined two important physiological thresholds during the water deficit period of P. sylvestris. The first threshold was obtained at−0.6 MPa and about 70% of stomatal closure (Fig.1a). The second threshold was observed at−1.8 MPa, where plants reached a zero net CO2uptake. The first, mild water deficit
phase (Ψpdrange from 0 to−0.6 MPa) was characterized by
sig-nificantreductionsinstomatalconductance(from500to150mmol
Fig. 1 Relationships of predawn shoot water potential (Ψpd) with
stomatal conductance (gs) (a) and with net CO2uptake (A) (b)
measured in five Scots pines along a water deficit period. I–III: physiological phases considered.Ψpd,1: limit between phases I and II
defined as the change in the slope of the relationship betweenΨpdand
gs.Ψpd,2: limit between phases II and III defined as the point where A is
zero
H2O m−2s−1) with moderate changes in photosynthesis rates over a
very narrow range ofΨpd(Fig.1). These physiological changes
would have induced at the end of phase I, a significant reduction of plant transpiration and a higher instantaneous water use efficiency. Additionally, the strong decrease in stomatal conductance over a very narrow range ofΨpdhas been reported previously in Pinus
radiata and interpreted as an evidence of strongly isohydric sto-matal response to water deficit (Brodribb and McAdam2013).
During the mild phase, the average values of all metabolite con-tents did not significantly differ from those in control trees.
4.2 Moderate soil water deficit induces changes in needle secondary metabolism
Phase II (−0.6 > Ψpd>−1.8 MPa)was characterized bystrongly
reduced stomatal conductance (below 150 mmol H2O m−2s−1)
Table 1 Average (± SE) concentrations (μg g−1) of monoterpenes, sesquiterpenoids, and resin acids of three control trees and five water deficiency-treated trees during three physiological phases (I–III)
Terpene Control trees Water-deficient trees
I II III
Ψpd>−0.6 −0.6 > Ψpd>−1.8 −1.8 > Ψpd>−2.4
Monoterpenes (μg g−1)
Tricyclene 21.4 ± 1.5a 23.3 ± 2.3a 35.3 ± 3.9b 44.9 ± 6.4b
α-Thujene 1.10 ± 0.09a 1.19 ± 0.12a 1.83 ± 0.17b 2.41 ± 0.31c
α-Pinene 275 ± 18a 266 ± 24a 456 ± 67b 528 ± 98b
Camphene 49.8 ± 3.9a 53.9 ± 5.6a 76.8 ± 69.0b 94.4 ± 13.0b
Sabinene 4.7 ± 0.5a 4.6 ± 0.6a 5.78 ± 0.72a 6.84 ± 1.02a
β-Pinene 29.0 ± 2.1a 47.1 ± 7.4ab 62.3 ± 8.6b 66.3 ± 14.6b
Myrcene 19.9 ± 1.8a 19.2 ± 2.2a 30.3 ± 3.4b 39.7 ± 5.5b
α-Phellandrene 0.49 ± 0.04a 0.44 ± 0.06a 0.84 ± 0.11b 1.15 ± 0.21b
Limonene 5.22 ± 0.50a 5.63 ± 0.71a 11.1 ± 1.52b 11.2 ± 1.92b
(Z)-β-Ocimene 0.57 ± 0.05a 0.55 ± 0.08a 1.23 ± 0.29b 1.07 ± 0.17b
(E)-β-Ocimene 5.66 ± 0.53a 7.55 ± 1.40ab 11.12 ± 1.52b 11.94 ± 2.25b
Terpinolene 0.39 ± 0.04a 0.42 ± 0.05a 1.23 ± 0.29b 1.29 ± 0.46b
Sesquiterpenoids (μg g−1)
β-Elemene 0.93 ± 0.08a 1.01 ± 0.11ab 1.46 ± 0.19bc 1.68 ± 0.28c
β-Caryophyllene 11.7 ± 0.5a 8.8 ± 0.6a 14.0 ± 1.9b 17.1 ± 2.5b
β-Copaene 0.078 ± 0.007a 0.072 ± 0.009a 0.20 ± 0.06b 0.15 ± 0.103ab
Aromadendrene 0.047 ± 0.005a 0.057 ± 0.015a 0.18 ± 0.04b 0.26 ± 0.081b
α-Humulene 5.01 ± 0.22a 4.25 ± 0.23a 7.23 ± 1.06b 8.70 ± 1.485b
γ-Muurolene 0.54 ± 0.05a 0.48 ± 0.06a 1.63 ± 0.53a 1.47 ± 0.95a
Germacrene D 20.7 ± 1.6a 16.0 ± 1.9a 26.2 ± 5.9a 20.7 ± 9.3a
β-Selinene 0.28 ± 0.02a 0.37 ± 0.09a 0.77 ± 0.14b 1.03 ± 0.283b
Bicyclogermacrene 0.25 ± 0.02a 0.23 ± 0.02a 0.51 ± 0.11b 0.60 ± 0.22b
α-Muurolene 0.72 ± 0.05a 0.71 ± 0.08a 0.99 ± 0.10b 1.04 ± 0.155b
γ-Cadinene 0.64 ± 0.05a 0.73 ± 0.09a 1.73 ± 0.40b 1.97 ± 0.678b
δ-Cadinene 0.42 ± 0.03a 0.54 ± 0.06a 1.26 ± 0.30b 1.54 ± 0.58b
GermacreneD-4-ol 0.54 ± 0.08a 0.62 ± 0.10a 0.95 ± 0.13b 0.94 ± 0.180b
α-Cadinol 0.15 ± 0.01a 0.25 ± 0.02a 0.42 ± 0.10a 0.43 ± 0.149a
Resin acids (μg g−1)
Pimaric acid 81 ± 8a 100 ± 11a 91 ± 10a 89 ± 17a
Isopimaric acid 22 ± 2a 24 ± 4a 22 ± 3a 23 ± 3a
Palustric acid 175 ± 18a 195 ± 38a 91 ± 17b 25 ± 8c
Levopimaric acid 513 ± 34a 606 ± 62a 405 ± 62a 152 ± 35b
Dehydroabietic acid 667 ± 43a 743 ± 102a 747 ± 77a 730 ± 84a
Abietic acid 183 ± 12a 205 ± 24a 178 ± 23a 138 ± 22a
Neoabietic acid 2007 ± 213a 2608 ± 469a 1495 ± 206b 523 ± 143c
Phases are defined according to changes in predawn water potential (Ψpd), stomatal conductance, and net assimilation rate (Fig.1). Compounds in each
and net CO2uptake rate (from 9 to 0μmolCO2m−2s−1) (Fig.1).
At this stage, trees started to accumulate most of the volatile terpenoids analyzed (monoterpenes and sesquiterpenoids). As stomatal aperture cannot significantly control the emissions of hydrophobic compounds (Niinemets and Reichstein2003), the accumulation of these volatile terpenes (although mostly stored in the resin ducts) cannot be explained by massive reductions in stomatal closure and rather suggests an increment in their syn-thesis. Such an increment has been reported in several cases in
plants treated with a water deficiency (Lluisà and Peñuelas
1998) and suggested to reflect changes in the source/sink bal-ance due to inhibition of growth (Lerdau et al.1994; Constable et al.1999). Indeed, this level of moderate soil water deficit usually substantially decreases the growth of conifers (e.g., Turtola et al.2003) and can lead to shifts in the translocation of the carbon fixed in photosynthesis to the formation of sec-ondary compounds (Lorio and Sommers1986; Turtola et al.
2003; McDowell2011; Niinemets2016). From an ecological perspective, greater contents of monoterpenes and sesquiterpenoids could enhance the tree resistance to herbivory and pathogens (Litvak and Monson1998; Cheng et al.2007).
Regarding resin acids, two of them, palustric and neoabietic acid, decreased analogously as the observations of Hodges and Lorio (1975) on Pinus taeda xylem oleoresin. Other studies had also reported variations in the concentration of wood resin acids with increasing the severity of water deficit (Turtola et al.2003) or an increase in resin acids in needles after the onset of a water stress decay process (Sanz et al.2014). However, to the best of our knowledge, no studies have reported a decrease in resin acids in conifer needles during a water deficit period. Such a decrease might reflect reprogramming of the defense metabolite pathway between syntheses of larger (resin acids) and smaller (monoterpenoids) plastidial isoprenoids, but so far, the way such metabolic regulation could occur is not known (Rajabi Memari et al.2013). Terpenoids are components of constitutive defenses against pests and pathogens with non-volatile resin acids involved in direct defenses (reducing palatability for her-bivores and sealing wound sites) (Hodges and Lorio 1975; Wainhouse 2004) and with more volatile monoterpenes and sesquiterpenoids involved in both direct (reducing palatability and serving as herbivore repellents) and indirect defenses
Fig. 2 Relationships between predawn shoot water potential (Ψpd) and
average (± SE) contents of proline (a), shikimic acid and total polyphenols (b), and abscisic acid (ABA) (c) in needles of five water-deficient Scots pines.Ψpd,1,Ψpd,2, and dotted lines indicate the limits of
the three physiological phases considered (I,Ψpd>−0.6 MPa; II,
−0.6 > Ψpd>−1.8 MPa; and III, −1.8 > Ψpd>−2.4 MPa)
Fig. 3 Relationship between the first two principal components (PC1 vs. PC2) of the principal component analysis (PCA) computed on needle metabolic compound variation. Symbols correspond to control (triangle) or water-deficient trees (circle) and are color-coded according to each physiological phase: I, white; II, gray; III, black
(signals for herbivore enemies) (Lewinsohn et al.1993; Litvak and Monson1998). Thus, changes in the composition of terpe-noids can importantly modify the needle capacity for direct and indirect defenses.
Finally, the slight increase of ABA observed at the end of phase II (Fig.2c) indicated the beginning of the contribution of ABA in stomatal closure. In order to reduce water loss in the plant, ABA affects guard cells by inducing osmotic efflux and therefore turgor loss and reduction of stomatal aperture. Stomatal closure detected before ABA concentration rising may respond only to the change in water potential at the leaf level (Buckley2005; Brodribb and McAdam2013).
4.3 Severe soil water deficit effects on needle secondary metabolism
Under severe soil water deficit, in phase III (−1.8>Ψpd>−2.4MPa)
characterized by minimum values of stomatal conductance and zero or close to zero net CO2uptake (Fig.1), there was a sudden increase
in the concentration of proline, shikimic acid, and ABA. The accu-mulation of proline in phase III confirms the suggestion that P. sylvestris was under a severe soil water deficit stress (Vilagrosa et al.2010). On the other hand, the carbon fixed during phase II under a moderate water deficit could also be used in phase III for synthesis of shikimic acid, one of the key sources of carbon for defense instead of growth (Becerra-Moreno et al.2015). Regarding the rapid increase in foliar ABA levels, Brodribb and McAdam (2013) suggested that such a major increase results in complete stomatal closure and drastic reduction of water loss. Both strategies, the increase of defense compounds and the preven-tion of desiccapreven-tion by stomatal closure, would benefit the tree during a short time period. If water deficit persists, with zero or close to zero photosynthetic carbon uptake and a continuous demand for carbo-hydrates to produce secondary compounds and to maintain metab-olism,thecarbohydratereserveswillbedepleted,leadingeventually to starvation or an inability to cope with biotic attacks (McDowell
2011). However, the time it takes to succumb to water deficit will ultimate depend on the size of the reserve pools (Niinemets2010). Nevertheless, contrary to shikimic acid, total polyphenol content was unaffected in the water potential range of our experiment. Given that polyphenols are derived from shikimic acid, and as the shikimic acid content did not change signifi-cantly until leaf water potential was less than−1.8 MPa, there is still a possibility that an increase in total needle polyphenol content could occur at more negative water potential values than the ones reached in this study. Alternatively, enhanced shikimic acid contents might reflect increased transformation of shikimic acid into lignin (Becerra-Moreno et al.2015) as observed in Pinus canariensis (Grill et al.2004). This could serve as a mechanical barrier against pathogens (Monties and Fukushima2001). In addition, more strongly lignified cell walls can enhance needle mechanical resistance avoiding col-lapse under excess water stress (Cochard et al.2004) and have
a greater change in water potential for a given change in nee-dle water content, thereby allowing for extraction of water from drier soil (Niinemets2001). Further studies looking into lignin formation and changes in needle water extraction ca-pacity are necessary to test these hypotheses.
Finally in phase III, the concentration of most volatile ter-penes that had increased significantly in phase II remained statistically constant, while the concentration of some resin acids (palustric, levopimaric, and neoabietic acid) kept de-creasing significantly, which might be a continuation of the reprogramming process of the defense metabolite pathway which started in phase II.
5 Conclusion
This study establishes a direct correspondence between phys-iological parameters and secondary metabolites through their simultaneous measurements along a soil water deficit period. Our results allowed the distinction of clearly physiological phases in response to water deficit evolution. In this way, a moderate water deficit (phase II) would be associated to in-creased levels of needle volatile terpenes with respect to phase I, whereas a severe water deficit (phase III) would be associ-ated to an increment in proline, shikimic acid, or ABA. Although there are biochemical processes related to secondary metabolism and plant-pathogen relations that are still poorly understood and deserve further investigations, this work con-firms that most of the secondary metabolites did not increase until the plant was under a moderate to severe water deficit.
Acknowledgements We thank Pilar Hijazo for the valuable help in the laboratory during the extraction and quantification processes of the met-abolic compounds.
Compliance with ethical standards
Funding Work of DSK is supported by a DOC INIA contract co-funded by INIA and ESF. This study was supported by Gobierno de Aragón (research group H38).
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